Tuberculosis is the leading cause of death in the world due to a single bacterial infection (27). Despite its enormous burden on world health, little is known about the molecular mechanisms of M. tuberculosis pathogenesis. Bacterial multiplication and concomitant tissue damage within an infected host, including experimentally infected mice, occurs primarily in the lungs--the favored niche of M. tuberculosis (28). Although it has been postulated that the distinctive cell wall of M. tuberculosis is important for virulence, rigorous genetic proof has been lacking. Using signature tagged mutagenesis, the inventors have isolated three attenuated M. tuberculosis mutants that are unable to synthesize or transport a complex, cell wall-associated lipid known as dimycoserosalphthiocerol (DIM). Two mutants of the present invention have transposon insertions affecting genes implicated in DIM synthesis, while the third mutant has a disruption of a gene encoding a large transmembrane protein required for DIM secretion. Surprisingly, synthesis and transport of this complex lipid is only required for growth in the lung; all three mutants are unaffected for growth in the liver and spleen. M. tuberculosis mutants deficient for DIM synthesis are attractive candidates for the development of a live, attenuated vaccine.